Enhancing laccase activity using pro-oxidants and pro-degradants

ABSTRACT

A two-component mediator system is disclosed, wherein one component serves as a pro-oxidant and the other component serves as a pro-degradant and booster. When used in combination with a laccase, the two-component mediators enhance the bleaching of pulp. In addition, a composition comprising an oxidative enzyme, a pro-oxidant, and a pro-degradant, and a process for oxidizing a substrate that comprises treating the substrate with an oxidizing enzyme, a pro-oxidant, and a pro-degradant are disclosed. The pro-oxidant can be ascorbic acid, ascorbate, salicylic acid, salicylate, nicotinic acid, nicotinate, a hardwood black liquor, a softwood black liquor, ligno-organosolv, lignin sulfonate, or mixtures thereof. The pro-degradant can be urea, thiourea, sulfamic acid, sulfamide, guanidine, methylsulfonic acid, or mixtures thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/318,294, filed on Sep. 10, 2001.

FIELD OF THE INVENTION

[0002] The present invention relates to the enhancement of enzymeactivity. More specifically, the present invention relates topro-oxidants and pro-degradants that, in combination, are useful inenhancing the activity of enzymes having laccase activity, especially inthe field of pulp bleaching.

BACKGROUND OF THE INVENTION

[0003] Paper pulp is typically processed from wood through the Kraft(and other) processes. The process produces a pulp with a dark browncolor, mostly due to the presence of lignin and lignin derivatives. Formany applications, the lignin has to be removed by a process known inthe art as “bleaching.” This is typically done commercially in severalstages in pulp mills, wherein lignin is first oxidized and then removedfrom the pulp.

[0004] Recently, several research groups have been working with enzymesto biologically bleach pulp, referred to as “bio-bleaching.”Bio-bleaching is a methodology whereby an enzyme is used to decrease theoptical brightness and/or lignin content of pulp or paper. The standardmeasure of bleaching efficiency is “Kappa number.” Enzymes that havemost commonly been used include laccase, lignin peroxidase, andmanganese peroxidase. An enzyme group that has received particularattention is the laccase family of enzymes, which are copper-containingenzymes that are known to be good oxidizing agents in the presence ofoxygen. Laccases are found in microbes, fungi, and higher organisms.

[0005] For many applications, the oxidizing efficiency of a laccase canbe improved through the use of a mediator, also known as an enhancingagent. Systems that include a laccase and a mediator are known in theart as laccase-mediator systems (LMS). There are several known mediatorsfor use in a laccase-mediator system. These include HBT(1-hydroxybenzotriazole), ABTS[2,2′-azinobis(3-ethylbenzothiazoline-6-sulfinic acid)], NHA(N-hydroxyacetinilide), NHAA (N-acetyl-N-phenylhydroxylamine), HBTO(3-hydroxy-1,2,3-benzotriazin-4(3H)-one), and VIO (violuric acid). Inaddition, there are several compounds containing NH—OH or N—O that havebeen found to be useful as mediators.

[0006] A major limitation of this methodology in the commercial contextis the cost and the efficiency of the mediators used. It is criticaltherefore to discover additional mediators.

SUMMARY OF THE INVENTION

[0007] The present invention provides two-component mediators, whereinone component serves as a pro-oxidant and the other component serves asa pro-degradant and booster. When used in combination with a laccase,the two-component mediators enhance the bleaching of pulp. Thus, theinvention provides a two-component mediator for use in alaccase-mediator system, the two-component mediator comprising (i) apro-oxidant and (ii) a pro-degradant.

[0008] The invention also provides a composition comprising an oxidativeenzyme, a pro-oxidant, and a pro-degradant The invention also provides aprocess for oxidizing a substrate that comprises treating the substratewith an oxidizing enzyme, a pro-oxidant, and a pro-degradant. Further,the invention provides a process for bleaching a lignin-containingmaterial that comprises treating the material with an oxidative enzyme,a pro-oxidant, and a pro-degradant.

[0009] The process of the invention can further include the step ofadding an oxidizing agent. In one embodiment, the oxidizing agent is atleast one of air, oxygen, and hydrogen peroxide.

[0010] In one embodiment, the pro-oxidant is ascorbic acid, ascorbate,salicylic acid, salicylate, nicotinic acid, nicotinate, a hardwood blackliquor, a softwood black liquor, ligno-organosolv, lignin sulfonate, ora mixture thereof. In another embodiment, the pro-degradant is urea,thiourea, sulfamic acid, sulfamide, guanidine, methylsulfonic acid, or amixture thereof.

DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention discloses the novel use of two componentsas mediators to enhance the bleach efficiency of an enzyme exhibitinglaccase activity. It has been discovered that, although the use of apro-oxidant or a pro-degradant alone as described herein is notnecessarily useful as a mediator, the combination of a pro-oxidant or apro-degradant together exert synergistic effects and produce betterbleach efficiency for paper pulp.

[0012] For ease of reference and without loss of generality, onecomponent is called a “pro-oxidant.” Examples of the pro-oxidant includeascorbic acid, ascorbate, salicylic acid, salicylate, nicotinic acid,nicoliante, hardwood black liquor, softwood black liquor,ligno-organosolv, lignin sulfonate, and mixtures thereof. Compounds withrelated structures or structural analogs may also be used. Thepro-oxidants are active mediators in the discoloration of a dye, e.g.,Chicago Blue. However, these may or may not have efficiency as mediatorsfor pulp bleaching.

[0013] For ease of reference and without loss of generality, the secondcomponent is called a “pro-degradant.” Examples of the pro-degradantinclude urea, thiourea, sulfamic acid, sulfamide, and guanidine.Compounds with related structures or structural analogs may also beused. The pro-degradants are often not active mediators, even in thediscoloration of a dye, e.g., Chicago Blue. In pulp bleaching tests withlaccase, they show either negative results or slightly positive results.

[0014] It has been discovered that when a pro-oxidant and apro-degradant are added together with a laccase enzyme, somecombinations are capable of a large decrease in the Kappa number. Theenzymes exhibiting laccase activity include the laccase enzymes ofenzyme classification EC 1.10.3.2, the catechol oxidase enzymes ofenzyme classification EC 1.10.3.1, the monophenol monooxygenase enzymesof enzyme classification EC 1.14.99.1, and the bilirubin oxidase enzymesof enzyme classification EC 1.3.3.5. The EC (Enzyme Commission) numberis based upon the Nomenclature Committee of the International Union ofBiochemistry and Molecular Biology (IUBMB).

[0015] The laccase of this invention may be derived from microbial,fungal, or other sources. It may furthermore be produced by arecombinant method, such as cultivating a host cell transformed with arecombinant DNA vector which includes a DNA sequence encoding thelaccase (and DNA sequences encoding functions that permit the expressionof laccase DNA sequence) in a culture medium under conditions thatpermit the expression of the laccase, and recovering the enzyme from theculture.

[0016] While the mediators are developed specifically for pulpbleaching, the same mediator mixtures and laccase enzyme(s) can be usedfor other applications, including treatment of pulp waste water,de-inking, industrial color removal, bleach for laundry detergents, oralcare teeth whiteners, and as catalysts or facilitators forpolymerization and oxidation reactions.

[0017] Another aspect of the invention provides a process for oxidizinga substrate that comprises treating the substrate with a compositioncomprising an oxidizing enzyme and a pro-oxidant and a pro-detergent.The pro-oxidant and pro-detergent can be selected from the abovedescribed pro-oxidants and pro-detergents.

[0018] The oxidative enzyme is a laccase, a catechol oxidase, amonophenol monooxygenase, a bilirubin oxidase, or a mixture thereof. Theprocess further comprises adding a hydrolase, such as xylanase.

[0019] The pro-oxidants and pro-detergents may each be individuallypresent in concentrations of from about 0.01 micromolar to 1000micromolar, more preferably from about 0.1 micromolar to about 250micromolar and most preferably from about 0.5 to about 100 micromolar.

[0020] The enzyme is used in amounts of from about 0.1 to 400 units(defined in Examples using ABTS as substrate) for 1 g dry pulp, morepreferably from 1 to 200 units and even more preferably from about 10 to100 units and most preferably from 20 to −50 units.

[0021] The process of the invention can further include the step ofadding an oxidizing agent, such as at least one of air, oxygen, andhydrogen peroxide.

[0022] One embodiment of the invention provides a process for bleachinga lignin-containing material that comprises treating the material withan oxidative enzyme and a pro-oxidant and a pro-detergent. In thisaspect of the invention, the a pro-oxidant and a pro-detergent may eachbe separately present in an amount of from about 0.1% to about 15% basedon the weight of the dry lignin containing material, more preferablyfrom about 0.1% to about 10% and even more preferably from about 0.5% toabout 5% and most preferably from about 1% to about 4%. The oxidativeenzyme is a laccase, a catechol oxidase, a monophenol monooxygenase, abilirubin oxidase, or a mixture thereof. In another embodiment, theprocess further comprises adding a hydrolase, such as xylanase.

[0023] One example of a lignin containing material is wood pulp. Theprocess for bleaching a lignin-containing material can further includethe step of adding an oxidizing agent, such as at least one of air,oxygen, and hydrogen peroxide.

EXAMPLES

[0024] Laccase Enzyme Assay

[0025] In the examples, two Aspergillus laccases have been used, bothfrom Novozymes A/S (Denmark). NovoSample 51002 works best at pH 4-5while NovoSample 51003 works best at pH 5-6. Enzyme dosage has beenfound to have an effect on bio-bleaching. For example, 0.1 ml of the51003 laccase gives a modest Kappa number reduction when HBT is used,and a huge reduction when ABTS is used.

[0026] The specific activity was determined using ABTS (0.5 mM) assubstrate. One unit of activity is equal to the umol of the oxidizedproduct from ABTS per min per mg protein at pH 6.0 at 23° C. Theextinction coefficient of the oxidized ABTS is: ε(max) at 420nm=36,000M-1 cm-1.)

[0027] Alternatively, the activity of laccase (NS51003) was determinedusing syringaldazine as substrate. In this case, one unit of activity isequal to the change of 0.001 UV absorbance at A530 nm per minute per ugprotein in 2 ml of 100 mM, pH 5.5 potassium phosphate buffer, and 0.5 mlof 0.25 mM syringaldazine in methanol at 23° C.

[0028] Kappa Number

[0029] Delignification of the pulp was measured as the change in Kappanumber according to TAPPI method T236 cm-85. Briefly, a known mass ofpaper pulp (containing lignin) was reacted with an excess of potassiumpermanganate in acid solution for a specified period of time to oxidizethe lignin. After the reaction, the residual permanganate was determinedby titration. The Kappa number was defined as the volume (ml) of 0.1Npotassium permanganate consumed by 1 g of moisture-free pulp in 0.5Nsulfuric acid after a ten-minute reaction time at 25° C. underconditions such that one-half of the permanganate remains unreacted. Alinear relationship with the lignin content of the pulp and themeasurement of the Kappa number has been done on samples as low as 300mg of pulp.

[0030] Pulp Bleaching

[0031] A softwood Kraft pulp, Kappa number 31.0, was treated with alaccase (NS51003) under the following conditions: Enzyme dosage 45units/g pulp pH 5.5 Temperature 50° C. Reaction time 16 hours Pulpconsistency 2%

[0032] The dried pulp was added to 80 ml of 50 mM phosphate, pH 5.5, anddisintegrated in a blender. The pulp was then transferred to a 500-mlconical flask and the blender was washed with 20 ml of the same buffer.The washed buffer and the pulp were combined. The mediator was added at1-4% (w/w, based on the dry pulp) followed by the addition of thelaccase. The pH of the pulp mixture was adjusted to 5.5 if needed. Theflask was covered with an aluminum foil with holes punched through andincubated at 50° C. for 16 hrs on a rotary shaker at 200 rpm.

[0033] After the enzymatic treatment, the pulp mixture was filteredthrough a Buchner funnel and the pulp was washed with water. The pulpfrom the control experiment was treated under the same pH andtemperature conditions as described above. The washed pulp was thentreated with an alkaline solution under the following conditions: Pulp 2g Water 200 ml NaOH 240 mg H₂O₂ (30%) 400 ul Temperature 70° C. Reactiontime 3 hours

[0034] The filtered pulp was repulped in the alkaline solution andincubated at 70° C. for 3 hrs. The pH of the pulp mixture should bebetween 11.7-12.00 during the entire treatment. After the treatment, thepulp mixture was filtered through a Buchner funnel and the pulp waswashed with water extensively and then dried in a hood overnight.

[0035] The delignification of the pulp was measured as the change in theKappa number according to TAPPI method T236 cm-85.

[0036] The following examples are illustrative of the present invention,and are not intended to be construed in any way as limiting the scope ofthe invention.

Example 1 Chicago Blue Assay of Pro-Oxidants and Pro-Degradants

[0037] In this example, the pro-oxidants and pro-degradants were testedindividually as mediators to enhance laccase-catalyzed bleaching ofChicago Blue in solution. The Chicago Blue Dye, also known as DirectBlue 1 or DB1, is a commonly used assay for the oxidative efficiency oflaccase. The Chicago Blue Dye is fully described by Schneider et al. inU.S. Pat. No. 5,885,304, which is hereby incorporated by reference.

[0038] Each of the compounds (i.e., potential mediators) was dissolvedin water, or in ethanol if the potential mediator was not water soluble,and then mixed with a phosphate buffer and a Chicago Blue solution. Asolution of a laccase was added to make up 1 ml of the final solution,containing 20 uM mediator, 20 mM buffer at pH 5.5 or 7.0, 0.1-1% laccase(v/v) and Chicago Blue solution, with absorbance at A610 nm between 0.6to 0.8. The change in the absorbance at A610 nm was measured immediatelyusing a UV-VIS spectrophotometer (UV-1201, Shimadzu ScientificInstruments) after the enzyme was added. The decrease in absorbance wasrecorded at 30-second intervals for 5 minutes and was used to estimatethe efficiency of the mediator.

[0039] The following are the results from the bleaching experiments ofChicago Blue: ΔmA610 Usage ΔmA610 (3 min) Mediator (0.1 umole/ml)(ugram) (3 min) at pH 5.5 at pH 7.0 none 0 0 ABTS 10 445 341 Ligninorganosolv 20 ug/ml 390 265 Ascorbic acid 20 ug/ml 6 2 Nicotinic acid 20ug/ml 0 0 Urea 20 ug/ml 1 0 Pine black liquor 100 (14%) 26 18Lignosulfonic acid 25 ug/ml 24 — Salicylic acid 20 ug/ml 11 —

Examples 2-6 Pulp Bleaching Using Pro-Oxidant and Pro-Degradant

[0040] In these examples, we used the pulp bleaching as described above,laccase as the oxidative enzyme, a pro-oxidant (as mediator 2), and apro-degradant (as mediator 1). Example laccase mediator 1 mediator 2pulp Decrease in Kappa 2 0 0 0 2 g 0 3 100 ul 0 0 2 g 2.6 4 100 ul urea,87 mg 0 2 g 2.6 5 100 ul 0 ascorbate, 20 mg 2 g 4.8 6 100 ul urea, 88 mgascorbate, 79 mg 2 g 7.1

[0041] The data above clearly indicate that urea is barely active inenhancing bleaching. However, in the presence of a pro-oxidant, anoticeable decrease in Kappa number was obtained.

Examples 7-13 Pulp Bleaching Using Pro-Oxidant and Pro-Degradant

[0042] In these further examples, we used the pulp bleaching asdescribed above, laccase as the enzyme, a pro-oxidant (as mediator 2),and a pro-degradant (as mediator 1). In the following table,SA=salicylic acid, AA=ascorbic acid. The decrease in Kappa number is thedifference in the Kappa numbers of the corresponding experiments withand without mediators under the same experimental conditions used forthe pulp tests. Laccase Pulp Decrease in Kappa Example (ml) Mediator 1Mediator 2 (g) number 7 0.1 SA, 80 mg Dicyandiamide, 80 mg 2 −4.7 8 0.1SA, 80 mg Melamine, 80 mg 2 −5.9 9 0.2 SA, 80 mg sulfanilic acid, 80 mg2 −4.0 10 0.15 SA, 80 mg Sulfanilamide, 80 mg 2 −3.3 11 0.2 AA, 80 mgSulfanilamide, 80 mg 2 −4.1 12 0.1 AA, 80 mg Melamine, 80 mg 2 −6.1 130.1 AA, 80 mg Dicyandiamide, 80 mg 2 −6.1

[0043] It is to be understood that the above described embodiments areillustrative only and that modification throughout may occur to oneskilled in the art. For example, a person of skill in the art willrecognize that the mediators of the invention also include mediatorswhich are functionally equivalent to the mediators specifically recitedherein, such equivalents having minor structural variations such as theaddition of a methyl or ethyl substituent or the formation of a methylester from a carboxylic acid. Accordingly, this invention is not to beregarded as limited to the embodiments disclosed herein.

What is claimed is:
 1. A process for bleaching a lignin-containingmaterial, comprising treating the material with an oxidative enzyme, apro-oxidant, and a pro-degradant.
 2. The process of claim 1 wherein saidoxidative enzyme is a laccase, a catechol oxidase, a monophenolmonooxygenase, a bilirubin oxidase, or a mixture thereof.
 3. The processof claim 1 further comprising adding a hydrolase.
 4. The process ofclaim 3 wherein said hydrolase is xylanase.
 5. The process of claim 1further comprising adding an oxidizing agent.
 6. The process of claim 5where said oxidizing agent is at least one of air, oxygen, and hydrogenperoxide.
 7. The process of claim 1 wherein said pro-oxidant is ascorbicacid, ascorbate, salicylic acid, salicylate, nicotinic acid, nicotinate,a hardwood black liquor, a softwood black liquor, ligno-organosolv,lignin sulfonate, or a mixture thereof.
 8. The process of claim 1 wheresaid pro-degradant is urea, thiourea, sulfamic acid, sulfamide,guanidine, methylsulfonic acid, or a mixture thereof.
 9. The process ofclaim 1 wherein said material is a wood pulp.
 10. The process of claim 9wherein said wood pulp is a raw material used to form a cellulose or acellulose derivative.
 11. A process for oxidizing a substrate, whereinsaid process comprises treating the substrate with an oxidizing enzyme,a pro-oxidant, and a pro-degradant.
 12. The process of claim 11 whereinsaid oxidizing enzyme is a laccase, a catechol oxidase, a monophenolmonooxygenase, a bilirubin oxidase, or a mixture thereof.
 13. Theprocess of claim 11 that further comprises adding an oxidizing agent.14. The process of claim 13 wherein said oxidizing agent is at least oneof air, oxygen, and hydrogen peroxide.
 15. The process of claim 11wherein said pro-oxidant is ascorbic, ascorbate, salicylic acid,salicylate, nicotinic acid, nicotinate, a hardwood black liquor, asoftwood black liquor, ligno-organosolv, lignin sulfonate, or a mixturethereof.
 16. The process of claim 11 wherein said pro-degradant is urea,thiourea, sulfamic acid, sulfamide, guanidine, methylsulfonic acid, or amixture thereof.
 17. The process of claim 11 that further comprisesadding an oxidizing agent.
 18. The process of claim 17 wherein saidoxidizing agent is at least one of air, oxygen, and hydrogen peroxide.19. A composition comprising an oxidative enzyme, a pro-oxidant, and apro-degradant.
 20. The composition of claim 19, wherein said oxidativeenzyme is a laccase, a catechol oxidase, a monophenol monooxygenase, abilirubin oxidase, or a mixture thereof.
 21. The composition of claim 19that further comprises a hydrolase.
 22. The composition of claim 21wherein said hydrolase is xylanase.
 23. The composition of claim 19wherein said pro-oxidant is ascorbic acid, ascorbate, salicylic acid,salicylate, nicotinic acid, nicotinate, a hardwood black liquor, asoftwood black liquor, ligno-organosolv, lignin sulfonate, or a mixturethereof.
 24. The composition of claim 19 wherein said pro-degradant isurea, thiourea, sulfamic acid, sulfamide, guanidine, methylsulfonicacid, or a mixture thereof.
 25. A two-component mediator for use in alaccase-mediator system, said two-component mediator comprising (i) apro-oxidant and (ii) a pro-degradant.
 26. The mediator of claim 25wherein: (i) said pro-oxidant is ascorbic acid, ascorbate, salicylicacid, salicylate, nicotinic acid, nicotinate, a hardwood black liquor, asoftwood black liquor, ligno-organosolv, lignin sulfonate, or a mixturethereof; and (ii) said pro-degradant is urea, thiourea, sulfamic acid,sulfamide, guanidine, methylsulfonic acid, or a mixture thereof.